The present invention relates to cryopreserved Composite Living Constructs (CCLCs) which are comprised of separated layers of cultured fibroblasts and cultured keratinocytes and to processes for making CCLCs. The CCLCs are prepared from composite living constructs (CLCs) by equilibrating with cryoprotectant solutions, freezing, and storing at cryogenic temperatures. Prior to use, they are thawed and rinsed to substantially remove the cryoprotectants.
CLCs are biologically active composite living constructs that are useful as wound dressings some of which comprise both a fibroblast dermal layer and an epidermal layer of keratinocytes usually on and/or in a matrix. CLCs may be employed for and aid in the regeneration of tissue in wounds that are denuded of skin such as granulating wounds, injuries such as those found in abrasions, excisions, burns and dystrophic epidermolysis bullosa and in ischemic skin such as that present in individuals suffering from decubitus, diabetic, and venous stasis ulcers.
The viability of CLCs as well as those of other cell constructs, such as skin equivalents, grafts, vessels, organs, etc., is short-lived unless such constructs are cryopreserved. Stability of stored cells and constructs are typically limited to about 24 to 120 hours unless they are protected from deterioration by some means such as cryopreservation. A primary goal of cryopreservation is to extend the storage stability of the CLCs without substantially compromising viability and metabolic activity as well as to permit them to maintain such viability and resume metabolic activity upon thawing and rinsing. xe2x80x9cStorage stabilityxe2x80x9d is the total time that a CCLC can be stored while minimally compromising viability and metabolic activity upon thawing and rinsing. Long storage stability is important for transplants or implants to be practical and commercially useful, to accommodate shipping and storage schedules, and to maintain sustainable inventories. Measurements useful in assessing the quality of the CCLC that is achieved by cryopreservation are: xe2x80x9cconstruct cell densityxe2x80x9d, the total number of viable cells per unit area; xe2x80x9ccell viabilityxe2x80x9d, the percent of the total number of cells that are viable; and xe2x80x9cmetabolic activityxe2x80x9d, a measure of the overall vigor of the viable cells in terms of their ability to metabolize nutrients and perform other cell maintenance functions. Additional measurements that may be applied to this invention are histologic examination of the structure of the CCLC for the presence, configuration, and distribution of cells within and on the construct, and the ability of the cells and constructs to express wound-healing and tissue regeneration promoters such as growth factors and cytokines.
Cryopreservation typically uses cryoprotectant solutions that comprise a buffered solution containing a non-cell-penetrating component, such as polysaccharides and glycosaminoglyans, e.g. dextran and chondroitin sulfate; and a cell-penetrating component; e.g., glycerol and dimethyl sulfoxide. It is suggested that the role of the cell-penetrating component is to limit cell-disrupting ice crystal formation and to limit destructive dehydration of the intracellular fluid; and that the role of the non-cell-penetrating component is to help maintain the physical integrity and architecture of the CLC.
It should be noted that the very processes of freezing and subsequent thawing inevitably cause a certain amount of cell loss and deterioration in cell viability and metabolic activity. This is due, in part, to the concentration and toxicity of the cryprotectants and the thermal shock and physical damages incurred during freezing and thawing. Thus, it is of importance to sustain the viability and metabolic activity of the thawed and rinsed product as closely as possible to that existing before cryopreservation.
Optimal formulation of cryoprotectants and conditions for crypreservation and for thawing and rinsing are dictated by the CLCs that are to be cryopreserved. The same cryopreservation approach cannot be applied to all CLCs. Formulations and process conditions depend on such factors as: cell type, construct configuration, mass, density, permeability and thickness. Such formulations and conditions include, for cryopreservation and long-term storage, the composition and program of addition and agitation of cryoprotectant solution and the freezing program and storage temperature. They additionally include, for preparation for use in humans and animals, requirements such as a thawing program and the composition of the rinse solution and a rinsing process.
This invention describes processes for cryopreserving such CLCs, and storing, thawing and rinsing such constructs to yield CCLCs that may, for example, be used to treat wounds in humans and animals. The CLCs employed in the processes of this invention are preferably comprised of separated cultured layers of human fibroblasts on and within a collagen sponge layer and human keratinocytes on a collagen layer that is semipermeable in that it is nonporous to biological cells and permeable to gases and to noncellular components. Such CLCs are described in U.S. Pat. Nos. 5,282,859, RE 35,399 and 6,039,859, to Eisenberg, the entire disclosures of which are incorporated herein by reference. Such CLCs are used, for example, as biologically active dressings for the treatment of acute and chronic skin wounds. The CLCs described in the foregoing Reissue Patent which are useful as starting CLCs in the present invention are composites comprising:
a) a porous sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,
b) a non-porous to cells, semipermeable second layer, which may or may not be in gel form, comprising a high purity collagen essentially free of exogenous glycosaminoglycans, said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and
c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer.
A collagen composite matrix useful as the structure for the fibroblasts and keratinocytes is described in claim 1 of U.S. Pat. No. 6,039,760 as follows:
A composite which comprises relative to a horizontal plane:
a) a first layer comprising a collagen matrix having upper and lower surfaces, said matrix being capable of permitting the growth of fibroblast cells therein, and being essentially non-contractible, and
b) a non-porous to cells, semipermeable second layer essentially free of exogenous glycosaminoglycans, said layer comprising a non-porous collagen layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and being sufficiently non-porous to be capable of maintaining on its surface keratinocyte cells without the substantial invasion of said cells into said collagen matrix.
Other CLCs may also be used as the starting materials in the cryopreservation process of the present invention. The process of the present invention is suitable for fibroblasts on or in any biocompatible absorbable porous material and keratinocytes on any biocompatible absorbent material. Such materials as collagen/GAG, crosslinked gelatin, alginates, synthetic absorbable polymers, such as polyglycolic acid, polycaprolactone co-polymers, and the like may be used.
It is an object of this invention to provide processes for making CCLCs having extended storage stability as well as acceptable cell viability, metabolic activity and structural integrity, compared to that of the corresponding CLC before cryopreservation.
A further object of this invention is to provide processes for making thawed and rinsed CCLCs having an acceptable cell viability, metabolic activity and structural integrity to be useful in treating wounds in humans and animals, compared to that of the corresponding CLCs before cryopreservation.
Still further objects of this invention are to provide CCLCs and thawed and rinsed CCLCs that result from the aforementioned processes.
Thus, embodiments of this invention encompass both the processes and the products therefrom that meet the foregoing objects.
One preferred embodiment of this invention provides a process for making a CCLC comprised of separated layers of cultured fibroblasts and keratinocytes, wherein the CCLC has a cell viability (i.e. the percent of total number of cultured fibroblasts and keratinocytes that are viable) of at least about 70% of the original and wherein the CCLC has a storage stability of at least about 6-12 months.
The CCLCs made by the processes of this invention comprise the following additional characteristics when compared with those of the CLC before cryopreservation: a total number of fibroblasts and keratinocytes which is at least about 50% of the original, a metabolic activity of the viable cells which is at least about 50% of the original cells, and a retention of structural integrity of the matrix.
The process of the invention comprises the steps of providing a CLC; equilibrating the CLC according to an equilibration program with a cryoprotectant solution comprising at least a non-cell penetrating component such as a polysaccharide or glycosaminoglycans, for example, dextran or chondroitin sulfate and preferably chondroitin sulfate and a cell-penetrating component such as glycerol or dimethyl sulfoxide and preferably dimethyl sulfoxide; lowering the temperature, according to a program, from about ambient temperature to about xe2x88x9290xc2x0 C.; and storing the CCLC at or below about xe2x88x92150xc2x0 C. The starting CLC which is subjected to the cryopreservation process of the present invention may be any CLC produced in any number of processes and of any composition provided it contains cells associated with a matrix which are to be cryopreserved. We prefer to use as the CLC starting material the composite prepared according to description in Eisenberg U.S. Pat. No. 5,282,859. In such a case, the invention contemplates providing a collagen matrix preferably comprised of a collagen sponge layer and a nonporous-to-cell, semipermeable collagen layer and seeding and culturing, in the presence of a cell growth medium, the fibroblasts on and within the collagen sponge and keratinocytes on the nonporous-to-cells, semipermeable collagen layer, thereby providing a CLC. Suitable results are obtained on CLCs about 6-7 cm per side and about 1-3 mm thick.
In the present invention, the CLCs are usually allowed to equilibrate with the cryoprotectant solutions at about ambient temperature after which the temperature is lowered at a rate optimal for the CLCs to slow or halt their cell metabolic activity and to achieve protection against dehydration while minimizing concomitant ice crystal formation. The temperature is lowered further to essentially halt metabolic activity, for long-term storage, to below about xe2x88x92150xc2x0 C. Thawing, usually to ambient temperature, is followed by rinsing the CCLC to substantially reduce cryoprotectant levels.
The equilibration program comprises the steps of: adding to the CLC a first solution containing the non-cell penetrating component, preferably chondroitin sulfate, at a basal concentration to form an initial equilibrated CLC (suitably between 2-3%), adding to the initial equilibrated CLC a second solution containing the cell-penetrating component, preferably dimethyl sulfoxide at an initial dimethyl sulfoxide concentration of about 20% (suitably between 18-22%) and containing chondroitin sulfate at the same basal concentration as in the first solution to form the final equilibrated CLC in which the chondroitin sulfate is maintained at the original chondroitin sulfate concentration and the dimethyl sulfoxide is brought to a final dimethyl sulfoxide concentration, preferably around 10% (suitably between 9-11%).
A second preferred embodiment of this invention provides a process for making a thawed and rinsed CCLC that may be used to treat wounds in humans and animals. The process for making a thawed and rinsed CCLC comprises, in addition to those given for making the CCLC, the steps of: warming the CCLC from the cold storage temperature to a first higher temperature of about xe2x88x92100xc2x0 C. by allowing it to stand at room temperature and then further warming the CCLC to a second higher temperature above 0xc2x0 C. (4xc2x0 C. to room temperature is suitable) by warming in room temperature saline or water and then rinsing the CCLC substantially free of cryoprotectants.